Preparing the sample for determination of Viability I. For preparing one sample for analysis you will need: - 1 pcs CELLCHIP - 1 piece of colored Eppendorf with SOFIA GREEN lyophilized dye - 1 piece of transparent Eppendorf with lyophilized SOFIA GREEN dye and lysis solution - 2 pcs of pipette tips - up to 100 μl - 2 pcs of pipette tips - up to 1000 μl - 2-4 pcs 15 ml tubes - 1 piece of 100 μl automatic pipette - 1 piece of 1000 μl automatic pipette - 1 piece Stirrer Mini vortex II. Principle of analysis The EASYCOUNTER YC-1 counts individual cells in suspension by detecting fluorescence signals of stained DNA in the cell nuclei. The staining dye used is SOFIA GREEN. The cell membrane of viable cells is virtually impermeable with regard to SOFIA GREEN, while membrane of non-viable, dead cells is permeable to SOFIA GREEN. The determination of the total concentration of cells requires that the membrane of viable cells is made permeable to SOFIA GREEN. This is achieved by mixing cell suspension with Lysis reagent. After Lysis reagent treatment (10 min), approximately 8 µl of the all cell stained with YO-DEDM 1 and 8 µl of the only dead cells stained with YO-DEDM 1 were loaded into the CELLCHIP. The CELLCHIP is placed in the EASYCOUNTER YC instrument where the cells are automatically enumerated. The accuracy of the measurement depends on the proper and consistent execution of all the stages of the sample preparation and the good stirring of the sample. To minimize difference in counting the results for multiple testing of the same sample, always stir the sample thoroughly before taking part of its content. The sample is representative only when it is well mixed. One very important factor is the rate of sample dilution. The concentration of the cell suspension to be transferred to the measuring chip and assayed must be within the optimum measuring range of the EASYCOUNTER YC apparatus. In order to provide a 46 EASYCOUNTER YC manual 2018 Milkotronic LTD
statistically reliable result, the total cell count in the diluted cell suspension should be in the range of 1x10 5 to 1x10 7 cells/ml. optimal range from 1х10 5 to 1х10 7 cells/ml measuring range - from 1х10 3 to 5х 10 7 cells/ml If cells concentration is in the range from 1x10 5 to 1x10 7 cells / ml, the initial sample is not diluted. If cell concentration is higher than 1x10 7 cells/ml the initial sample is diluted with distilled water. The rate of sample dilution for determination of dead cell count is 10 times lower than the rate of dilution for determination of total cell count; this is due to the fact that dead cells concentration in the sample is usually much lower than total cell concentration. III. Determination of dilution rate А. In case of unknown sample cell concentration 1. Take a 15 ml centrifuge tube containing 10 ml homogenized sample. 2. Place the tube in a centrifuge and balance the rotor with an opposing tube as necessary. 3. Centrifuge for 6 minutes at 4000 rpm. 4. Remove the test tube being careful not to disturb the sediment. 5. Measure the total volume in the tube, the volume of supernatant and from the difference - volume of sediment. 6. Calculate the percentage of sedimentation by the following formula: % sediment = sediment volume/total volume х 100 7.. Determine the rate of sample dilution according to Table 1, depending on the determined % sediment. B. With known indicative sample cell concentration Usually, in beer, wine and bioethanol production it is known in what range cell concentration varies at different stages of production. In this case, it is not necessary to determine % spun solids, but the sample is directly diluted with distilled water. Table 1 shows how many times the initial sample is diluted according to cell concentration in the sample. The rate of sample dilution for determination of dead cell count is 10 times lower than the rate of dilution for determination of total cell count, 47 EASYCOUNTER YC manual 2018 Milkotronic LTD
this is due to the fact that dead cells concentration in the sample is usually much lower than total cell concentration. Table 1. Dilution of the cell suspension depending on the initial cell concentration in order to reach the optimal measuring interval for total and dead cell count determination. Spun solids Total number of cells in the initial sample, cells/ml Dilution of the initial sample, how many times for a total number of cells (transparent eppendorf) for dead cells (colored eppendorf) 80% 1500-2500 х 10 6 1000 100 50-80% 500-1500 х 10 6 500 50 5-50% 100 500 х 10 6 100 10 5% 100 х 10 6 20 2 If the concentration of the cells in the undiluted sample is below 1x10 5 cells / ml, the concentration should be increased by centrifugation (> 10000xg, 15min) with subsequent resuspension of the pellets with PBS IV. Measurement Procedure 1. Take two tubes (15 ml volume) and place in each of them 1 ml of the cell suspension using an automatic pipette. Add distilled water in order to achieve optimal dilution (Table 1), according to the indicative initial cell concentration. The sample in the first tube is diluted and used for determination of dead cell count. Тhe sample in the second tube is diluted 10-times more than first tube and it is used for determination of total cell count. When suctioning the cell suspension, hold the tip over the bottom of the tubes and observe particles from the medium not to impede the suction of the liquid sample. Even a partial occlusion of the tip entry may result in a reduction in the number of cells in the analyzed sample. 48 EASYCOUNTER YC manual 2018 Milkotronic LTD
If the cell suspension is with high density (sample from the propagator in brewing production), weigh 1 gram of suspension in each of the two 15 ml tubes at analytical balance using a special polystyrene foam stand and dilute with distilled water, 1000 times to determine the total cell count and 100 times to determine the dead cell count. Always indicate the rate of dilution in the tubes to know which tube to take when determining the dead cells and which ones to determine the total number of cells! 2. Take the two test tubes from the stand and homogenise them by placing the tip of each of them in the Mini Vortex stirrer opening. Press and hold the tip for 5 seconds and pull back. Repeat 8-9 times. (see 1.1, 1.2 and 1.3) 1.1 1.2 1.3 3. Take 1 ml of the diluted and homogenized sample in the 15 ml tube intended for determination of total cell count and put it in the transparent microcentrifuge tube (Eppendorf) that contains lyophilized lysis reagent and fluorescent dye SOFIA GREEN. 4. Take 1 ml of the diluted and homogenized sample in the 15 ml tube intended for determination of dead cell count and put it in the colored microcentrifuge tube (Eppendorf) that contains lyophilized fluorescent dye SOFIA GREEN.. Close both the tubes. 5. Take the transparent microcentrifuge tube from the stand and place the tip in the Mini Vortex stirrer opening. Press and hold the tip for 5 seconds and then pull it back. Repeat 8-9 times. Repeat this procedure for the other microtube (see 2.1, 2.2, 2.3). The transparent tube contains a lot of analytes and it takes more time to dissolve. Stir until complete dissolution of the dry analyte. The transparent tube contains a lot of analytes and it takes more time to dissolve. Stir until complete dissolution of the dry analyte 49 EASYCOUNTER YC manual 2018 Milkotronic LTD
2.1 2.2 2.3 6. Incubate both the samples for 10 min to let the sample interact with the lysis reagent and the fluorescent dye SOFIA GREEN. 7. Mix both the samples again using Mini Vortex. Press and hold the tip for 5 seconds and then pull it back. Repeat 2-3 times (see 2.1, 2.2 and 2.3). 8. Unpack one CELLCHIP. Do not touch the upper surface of the CELLCHIP. Always hold it on the side edges. 9. Load the two samples in the CELLCHIP microfluidic chambers by the 8 μl pre-set automatic pipette. In the microfluidic chamber A, place 8 μl of a sample from the transparent microcentrifuge tube (for total cell count) and 8 μl sample from the colored microcentrifuge tube (for dead cells) in microfluidic chamber B. Pipette the solution at an angle of approximately 80 to the semi-circle loop hole. Pipetting is done by gently pushing the pipette button from start position to first stop (see 3.1, 3.2). Hold the first stop button, withdraw the pipette from CELLCHIP and then slowly release the button to the initial position (see 3.3). Do not use the second stop to prevent air from entering the microfluidic chamber. In this way, you will completely empty the tip and ensure precise pipetting. Always dispose of used tip and use a new tip for the next sample Avoid the formation of air bubbles in the microfluidic chamber and reverse splashes during 50 EASYCOUNTER YC manual 2018 Milkotronic LTD
3.1 3.2 3.3 To load the rest two chambers C and D, for measuring another yeast sample repeat the procedure described in point 1 to 9. Follow the sequence: the sample from the transparent microcentrifuge tube should be loaded in chamber C and the sample from the colored microcentrifuge tube in chamber D. 10. Place the loaded CELLCHIP in EASTCOUNTER YC cartridge. 11. Using the software proceed to sample analysis. It is recommended to place the loaded CELLCHIP in the unit and start the analysis within 30-60 seconds. If the sample remains in the CELLCHIP in the apparatus longer, when you start the analysis it may result in inaccurate results due to evaporation of the sample and getting air into it. V. Disposal of waste: Using the automatic pipette second button, remove the tip into a suitable container. Discard the microfuge tube with the remaining sample, the tips, and CELLCHIP in a suitable container. VI. Storage of EASTCOUNT YC KIT: EASTCOUNT YC KIT should be stored at -10 C to +40 C and short-term to -20 C, protected from direct sunlight. VII. Application: Working with an automatic pipette Before start operating the automatic pipettes, read the Instructions for Use of Automatic Pipette carefully. Make few attempts to suck in and suck out water to sense when you reach the first and second stops of the working button. Review the pipette manual video in the YOUTUBE at address: www.youtube.com/autocellcount. 51 EASYCOUNTER YC manual 2018 Milkotronic LTD
Take the automatic pipette pre-set to a specific volume. Make sure the front pipette cone is clean. Place it vertically over a tip from the tips arranged on the work stand and put in the pipette tip by gently pressing. From the initial position (see 4.1), press the pipette button until the first stop (see 4.2), hold it down and immerse the 2-3 mm tip of the pipette into the solution (see 4.3). Gently push out the operational button and remove the pipette from the liquid by touching the walls of the tube to remove excess yeast suspension (see 4.4, 4.5). 4.1 4.2 4.3 4.4 4.5 Pipette the yeast suspension from the tip into the open micro tube of the tripod by gently tapping the pipette button from the initial position to the first stop (see 4.6, 4.7). After a short pause, press the button next to the second stop (see 4.8). In this way, you will completely empty the tip and ensure precise pipetting. 4.6 4.7 4.8 4.9 For more information and video instructions for working with EASTCOUNT YC KIT, visit www.autocellcount.com or www.youtube.com/autocellcount 52 EASYCOUNTER YC manual 2018 Milkotronic LTD